nadE

ABSTRACT

The invention provides nadE polypeptides and polynucleotides encoding nadE polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing nadE polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the NAD biosynthesisfamily, as well as their variants, herein referred to as “nadE,” “nadEpolynucleotide(s),” and “nadE polypeptide(s)” as the case may be.

BACKGROUND OF THE INVENTION

[0002] The Streptococci make up a medically important genera of microbesknown to cause several types of disease in humans, including, forexample, otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid. Since its isolation more than 100 years ago, Streptococcuspneumoniae has been one of the more intensively studied microbes. Forexample, much of our early understanding that DNA is, in fact, thegenetic material was predicated on the work of Griffith and of Avery,Macleod and McCarty using this microbe. Despite the vast amount ofresearch with S. pneumoniae, many questions concerning the virulence ofthis microbe remain. It is particularly preferred to employStreptococcal genes and gene products as targets for the development ofantibiotics.

[0003] The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant stains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains that are resistantto some or all of the standard antibiotics. This phenomenon has createdan unmet medical need and demand for new anti-microbial agents,vaccines, drug screening methods, and diagnostic tests for thisorganism.

[0004] Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

[0005] Clearly, there exists a need for polynucleotides andpolypeptides, such as the nadE embodiments of the invention, that have apresent benefit of, among other things, being useful to screen compoundsfor antimicrobial activity. Such factors are also useful to determinetheir role in pathogenesis of infection, dysfunction and disease. Thereis also a need for identification and characterization of such factorsand their antagonists and agonists to find ways to prevent, ameliorateor correct such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

[0006] The present invention relates to nadE, in particular nadEpolypeptides and nadE polynucleotides, recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, includingtreatment of microbial diseases, amongst others. In a further aspect,the invention relates to methods for identifying agonists andantagonists using the materials provided by the invention, and fortreating microbial infections and conditions associated with suchinfections with the identified agonist or antagonist compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with microbial infections and conditionsassociated with such infections, such as assays for detecting nadEexpression or activity.

[0007] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

DESCRIPTION OF THE INVENTION

[0008] The invention relates to nadE polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a nadE of Streptococcuspneumoniae, that is related by amino acid sequence homology toNADE₁₃ECOLI NH(3)-DEPENDENT NAD(+) SYNTHETASE

[0009] (NITROGEN-REGULATORY PROTEIN) polypeptide. The invention relatesespecially to nadE having a nucleotide and amino acid sequences set outin Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively. Note thatsequences recited in the Sequence Listing below as “DNA” represent anexemplification of the invention, since those of ordinary skill willrecognize that such sequences can be usefully employed inpolynucleotides in general, including ribopolynucleotides. TABLE 1 NadEPolynucleotide and Polypeptide Sequences (A) Streptococcus pneumoniaenadE polynucleotide sequence [SEQ ID NO:1]. 5′-ATGAGTTTGCAAGAAACGATTATCCAAGAGCTGGGTGTCAAACCAGTGATTGATGCCCAGGAAGAAATCCGTCGTTCTATTGATTTCTTAAAAAGATATCTGAAAAAACATCCCTTCCTAAAAACCTTTGTACTAGGGATTTCTGGGGGACAAGACTCAACCTTGGCAGGACGTTTGGCGCAATTAGCTATGGAAGAACTGCGAGCTGAAACGGGAGACGATAGCTACAAATTTATCGCTGTCCGCCTGCCATACGGAGTGCAAGCTGATGAAGCAGATGCTCAAAAAGCCCTAGCCTTCATCCAGCCAGATGTCAGCTTGGTTGTGAATATCAAGGAATCAGCTGATGCCATGACAGCTGCAGTTGAAGCGACAGGTAGTCCTGTTTCAGACTTCAACAAGGGGAATATCAAGGCACGTTGCCGTATGATTGCTCAGTATGCCCTTGCTGGTTCCCATAGCGGAGCGGTCATTGGAACAGACCACGCCGCAGAAAATATCACAGGTTTCTTTACCAAGTTTGGTGACGGCGGTGCGGATATTCTCCCTCTTTACCGCCTCAATAAACGCCAAGGAAAACAGCTCTTGCAGAAACTTGGCGCAGAGCCAGCCCTTTATGAAAAAATCCCAACGGCAGACCTAGAAGAAGATAAACCAGGCCTAGCTGACGAAGTCGCACTTGGAGTCACCTACGCAGAGATTGACGACTACCTAGAAGGCAAAACAATCAGCCCAGAAGCTCAAGCGACCATTGAAAACTGGTGGCACAAAGGCCAACACAAACGCCACTTACCCATCACCGTATTTGATGACTTTTGGGAGTAA-3′ (B)Streptococcus pneumoniae nadE polypeptide sequence deduced from apolynucleotide sequence in this table [SEQ ID NO:2]. NH₂-MSLQETIIQELGVKPVIDAQEEIRRSIDFLKRYLKKHPFLKTFVLGISGGQDSTLAGRLAQLAMEELRAETGDDSYKFIAVRLPYGVQADEADAQKALAFIQPDVSLVVNIKESADAMTAAVEATGSPVSDFNKGNIKARCRMIAQYALAGSHSGAVIGTDHAAENITGFFTKFGDGGADILPLYRLNKRQGKQLLQKLGAEPALYEKIPTADLEEDKPGLADEVALGVTYAEIDDYLEGKTISPEAQATIENWWHKGQHKRHLPITVFDDFWE-COOH

[0010] Deposited materials

[0011] A deposit comprising a Streptococcus pneumoniae 0100993 strainhas been deposited with the National Collections of Industrial andMarine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB21RY, Scotland on 11 Apr. 1996 and assigned deposit number 40794. Thedeposit was described as Streptococcus pneumoniae 0100993 on deposit. On17 Apr. 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coliwas similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

[0012] The deposited strain comprises a full length nadE gene. Thesequence of the polynucleotides comprised in the deposited strain, aswell as the amino acid sequence of any polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

[0013] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The depositedstrain will be irrevocably and without restriction or condition releasedto the public upon the issuance of a patent. The deposited strain isprovided merely as convenience to those of skill in the art and is notan admission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

[0014] In one aspect of the invention there is provided an isolatednucleic acid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is comprisedin the deposited strain. Further provided by the invention are nadEpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare nadE polypeptide and polynucleotide sequences isolated from thedeposited

[0015] Polypeptides

[0016] NadE polypeptide of the invention is substantiallyphylogenetically related to other proteins of the NAD biosynthesisfamily.

[0017] In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as “nadE” and “nadEpolypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0018] Among the particularly preferred embodiments of the invention arevariants of nadE polypeptide encoded by naturally occurring alleles of anadE gene.

[0019] The present invention further provides for an isolatedpolypeptide that: (a) comprises or consists of an amino acid sequencethat has at least 95% identity, most preferably at least 97-99% or exactidentity, to that of SEQ ID NO: 2 over the entire length of SEQ ID NO:2; (b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence that has at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO: 1over the entire length of SEQ ID NO: 1; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO: 2, over the entire length of SEQ ID NO: 2.

[0020] The polypeptides of the invention include a polypeptide of Table1 [SEQ ID NO: 2] (in particular a mature polypeptide) as well aspolypeptides and fragments, particularly those that has a biologicalactivity of nadE, and also those that have at least 95% identity to apolypeptide of Table 1 [SEQ ID NO: 2] and also include portions of suchpolypeptides with such portion of the polypeptide generally comprisingat least 30 amino acids and more preferably at least 50 amino acids.

[0021] The invention also includes a polypeptide consisting of orcomprising a polypeptide of the formula:

X—(R ₁)_(m)—(R ₂)—(R ₃)_(n—) Y

[0022] wherein, at the amino terminus, X is hydrogen, a metal or anyother moiety described herein for modified polypeptides, and at thecarboxyl terminus, Y is hydrogen, a metal or any other moiety describedherein for modified polypeptides, R₁ and R₃ are any amino acid residueor modified amino acid residue, m is an integer between 1 and 1000 orzero, n is an integer between 1 and 1000 or zero, and R₂ is an aminoacid sequence of the invention, particularly an amino acid sequenceselected from Table 1 or modified forms thereof. In the formula above,R₂ is oriented so that its amino terminal amino acid residue is at theleft, covalently bound to R₁, and its carboxy terminal amino acidresidue is at the right, covalently bound to R₃. Any stretch of aminoacid residues denoted by either R₁ or R₃, where m and/or n is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer. Other preferred embodiments of the invention are providedwhere m is an integer between 1 and 50, 100 or 500, and n is an integerbetween 1 and 50, 100, or 500.

[0023] It is most preferred that a polypeptide of the invention isderived from Streptococcus pneumoniae, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0024] A fragment is a variant polypeptide having an amino acid sequencethat is entirely the same as part but not all of any amino acid sequenceof any polypeptide of the invention. As with nadE polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region in a single larger polypeptide.

[0025] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NO: 2], orof variants thereof, such as a continuous series of residues thatincludes an amino- and/or carboxyl-terminal amino acid sequence.Degradation forms of the polypeptides of the invention produced by or ina host cell, particularly a Streptococcus pneumoniae, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

[0026] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2,or an isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO: 2.

[0027] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention

[0028] Polynucleotides

[0029] It is an object of the invention to provide polynucleotides thatencode nadE polypeptides, particularly polynucleotides that encode apolypeptide herein designated nadE.

[0030] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding nadE polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO: 1] that includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Streptococcus pneumoniae.

[0031] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing nadE polypeptides andpolynucleotides, particularly Streptococcus pneumoniae nadE polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

[0032] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes anadE polypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNO: 2] and polynucleotides closely related thereto and variants thereof.

[0033] In another particularly preferred embodiment of the inventionthere is a nadE polypeptide from Streptococcus pneumoniae comprising orconsisting of an amino acid sequence of Table 1 [SEQ ID NO: 2] or avariant thereof.

[0034] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NO: 1], a polynucleotide of theinvention encoding nadE polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Streptococcus pneumoniae0100993 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a polynucleotide sequence given in Table 1 [SEQ IDNO: 1], typically a library of clones of chromosomal DNA ofStreptococcus pneumoniae 0100993 in E. coli or some other suitable hostis probed with a radiolabeled oligonucleotide, preferably a 17-mer orlonger, derived from a partial sequence. Clones carrying DNA identicalto that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynucleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO: 1] was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

[0035] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO: 1]contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO: 2] with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO: 1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 823 of SEQ ID NO: 1, encodes thepolypeptide of SEQ ID NO: 2.

[0036] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of: (a) apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO: 1 over theentire length of SEQ ID NO: 1; (b) a polynucleotide sequence encodng apolypeptide that has at least 95% identity, even more preferably atleast 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO: 2,over the entire length of SEQ ID NO: 2.

[0037] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO: 1 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

[0038] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in Table 1[SEQ ID NO: 1]. Also provided by the invention is a coding sequence fora mature polypeptide or a fragment thereof by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0039] A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 823 set forth in SEQ ID NO: 1 ofTable 1, both of that encode a nadE polypeptide.

[0040] The invention also includes a polynucleotide consisting of orcomprising a polynucleotide of the formula:

X—(R ₁)_(m)—(R ₂)—(R₃)_(n—) Y

[0041] wherein, at the 5′ end of the molecule, X is hydrogen, a metal ora modified nucleotide residue, or together with Y defines a covalentbond, and at the 3′ end of the molecule, Y is hydrogen, a metal, or amodified nucleotide residue, or together with X defines the covalentbond, each occurrence of R₁ and R₃ is independently any nucleic acidresidue or modified nucleic acid residue, m is an integer between 1 and3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is anucleic acid sequence or modified nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1 ora modified nucleic acid sequence thereof. In the polynucleotide formulaabove, R₂ is oriented so that its 5′ end nucleic acid residue is at theleft, bound to R₁, and its 3′ end nucleic acid residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either R₁and/or R₂, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Where, in apreferred embodiment, X and Y together define a covalent bond, thepolynucleotide of the above formula is a closed, circularpolynucleotide, that can be a double-stranded polynucleotide wherein theformula shows a first strand to which the second strand iscomplementary. In another preferred embodiment m and/or n is an integerbetween 1 and 1000. Other preferred embodiments of the invention areprovided where m is an integer between 1 and 50, 100 or 500, and n is aninteger between 1 and 50, 100, or 500.

[0042] It is most preferred that a polynucleotide of the invention isderived from Streptococcus pneumoniae, however, it may preferably beobtained from other organisms of the same taxonomic genus. Apolynucleotide of the invention may also be obtained, for example, fromorganisms of the same taxonomic family or order.

[0043] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae nadEhaving an amino acid sequence set out in Table 1 [SEQ ID NO: 2]. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

[0044] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO: 2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

[0045] Further particularly preferred embodiments are polynucleotidesencoding nadE variants, that have the amino acid sequence of nadEpolypeptide of Table 1 [SEQ ID NO: 2] in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,modified, deleted and/or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of nadE polypeptide.

[0046] Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO: 1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO: 1.

[0047] Further preferred embodiments of the invention arepolynucleotides that are at least 95% or 97% identical over their entirelength to a polynucleotide encoding nadE polypeptide having an aminoacid sequence set out in Table 1 [SEQ ID NO: 2], and polynucleotidesthat are complementary to such polynucleotides. Most highly preferredare polynucleotides tat comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

[0048] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1].

[0049] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to nadE polynucleotidesequences, such as those polynucleotides in Table 1.

[0050] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1× SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0051] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library comprising a complete gene for a polynucleotidesequence set forth in SEQ ID NO: 1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO: 1 or a fragment thereof; and isolatingsaid polynucleotide sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers fully describedelsewhere herein.

[0052] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding nadE and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to a nadE gene. Suchprobes generally will comprise at least 15 nucleotide residues or basepairs. Preferably, such probes will have at least 30 nucleotide residuesor base pairs and may have at least 50 nucleotide residues or basepairs. Particularly preferred probes will have at least 20 nucleotideresidues or base pairs and will have lee than 30 nucleotide residues orbase pairs.

[0053] A coding region of a nadE gene may be isolated by screening usinga DNA sequence provided in Table 1 [SEQ ID NO: 1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

[0054] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the DNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0055] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0056] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of Table 1 [SEQ ID NOS: 1 or 2] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0057] The invention also provides polynucleotides that encode apolypeptide that is a mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to a maturepolypeptide (when a mature form has more than one polypeptide chain, forinstance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away froma mature protein by cellular enzymes.

[0058] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0059] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0060] As will be recognized, the entire polypeptide encoded by an openreading frame is often not required for activity. Accordingly, it hasbecome routine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO: 1 readily provides contiguous fragments of SEQ ID NO:2 sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO: 2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

[0061] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps tat produce active and mature forms of the polypeptide.

[0062] Vectors, Host Cells, Expression Systems

[0063] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0064] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

[0065] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal.,BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0066] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococci E.coli, streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

[0067] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

[0068] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0069] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0070] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0071] This invention is also related to the use of nadE polynucleotidesand polypeptides of the invention for use as diagnostic reagents.Detection of nadE polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a human, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the nadE gene or protein, may bedetected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

[0072] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled nadE polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. NatlAcad. Sci., USA, 85: 4397-4401 (1985).

[0073] In another embodiment, an array of oligonucleotides probescomprising nadE nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al, Science, 274: 610 (1996)).

[0074] Thus in another aspect, the present invention relates to adiagnostic kit that comprises: (a) a polynucleotide of the presentinvention, preferably the nucleotide sequence of SEQ ID NO: 1, or afragment thereof; (b) a nucleotide sequence complementary to that of(a); (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO: 2 or a fragment thereof, or (d) an antibody toa polypeptide of the present invention, preferably to the polypeptide ofSEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c)or (d) may comprise a substantial component. Such a kit will be of usein diagnosing a disease or susceptibility to a Disease, among others.

[0075] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO: 1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0076] The differences in a polynucleotide and/or polypeptide sequencebetween organisms possessing a first phenotype and organisms possessinga different, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

[0077] Cells from an organism carrying mutations or polymorphisms(allelic variations) in a polynucleotide and/or polypeptide of theinvention may also be detected at the polynucleotide or polypeptidelevel by a variety of techniques, to allow for serotyping, for example.For example, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to use RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR As an example, PCRprimers complementary to a polynucleotide encoding nadE polypeptide canbe used to identify and analyze mutations. The invention furtherprovides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′and/or the 3′ end. These primers may be used for, among other things,amplifying nadE DNA and/or RNA isolated from a sample derived from anindividual, such as a bodily material. The primers may be used toamplify a polynucleotide isolated from an infected individual, such thatthe polynucleotide may then be subject to various techniques forelucidation of the polynucleotide sequence. In this way, mutations inthe polynucleotide sequence may be detected and used to diagnose and/orprognose the infection or its stage or course, or to serotype and/orclassify the infectious agent.

[0078] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1]. Increased or decreased expression of a nadE polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

[0079] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of nadE polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a nadE polypeptide, in a sample derived from a host, such as abodily material, are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis, antibody sandwich assays, antibody detection and ELISAassays.

[0080] Antagonists and Agonists - Assays and Molecules

[0081] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

[0082] Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g., inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofnadE polypeptides and polynucleotides; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

[0083] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists, in the absence of an agonist orantagonist, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution comprising apolypeptide or polynucleotide of the present invention, to form amixture, measuring nadE polypeptide and/or polynucleotide activity inthe mixture, and comparing the nadE polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and nadE polypeptide, as herein described, can alsobe used for high-throughput screening assays to identify antagonists ofthe polypeptide of the present invention, as well as of phylogeneticallyand and/or functionally related polypeptides (see D. Bennett et al., JMol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

[0084] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents that may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0085] The invention also provides a method of screening compounds toidentify those that enhance (agonist) or block (antagonist) the actionof nadE polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising nadE polypeptide and a labeled substrate or ligandof such polypeptide is incubated in the absence or the presence of acandidate molecule that may be a nadE agonist or antagonist. The abilityof the candidate molecule to agonize or antagonize the nadE polypeptideis reflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of nadE polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in nadE polynucleotide or polypeptideactivity, and binding assays known in the art.

[0086] Polypeptides of the invention may be used to identify membranebound or soluble receptors, if any, for such polypeptide, throughstandard receptor binding techniques known in the art. These techniquesinclude, but are not limited to, ligand binding and crosslinking assaysin which the polypeptide is labeled with a radioactive isotope (forinstance, ¹²⁵I), chemically modified (for instance, biotinylated), orfused to a peptide sequence suitable for detection or purification, andincubated with a source of the putative receptor (e.g., cells, cellmembranes, cell supernatants, tissue extracts, bodily materials). Othermethods include biophysical techniques such as surface plasmon resonanceand spectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

[0087] The fluorescence polarization value for a fluorescently-taggedmolecule depends on the rotational correlation time or tumbling rate.Protein complexes, such as formed by nadE polypeptide associating withanother nadE polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently-labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

[0088] Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of nadE polypeptide dimers,trimers, tetramers or higher order structures, or structures formed bynadE polypeptide bound to another polypeptide. NadE polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

[0089] Surface plasmon resonance can be used to monitor the effect ofsmall molecules on nadE polypeptide self-association as well as anassociation of nadE polypeptide and another polypeptide or smallmolecule. nadE polypeptide can be coupled to a sensor chip at low sitedensity such that covalently bound molecules will be monomeric. Solutionprotein can then passed over the nadE polypeptide -coated surface andspecific binding can be detected in real-time by monitoring the changein resonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for nadE polypeptideself-association as well as an association of nadE polypeptide andanother polypeptide or small molecule.

[0090] A scintillation proximity assay may be used to characterize theinteraction between an association of nadE polypeptide with another nadEpolypeptide or a different polypeptide . nadE polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled nadEpolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon nadE polypeptide binding and compounds that prevent nadEpolypeptide self-association or an association of nadE polypeptide andanother polypeptide or small molecule will diminish signal.

[0091] In other embodiments of the invention there are provided methodsfor identifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

[0092] Another example of an assay for nadE agonists is a competitiveassay that combines nadE and a potential agonist with nadE-bindingmolecules, recombinant nadE binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. nadE can be labeled, such as byradioactivity or a calorimetric compound, such that the number of nadEmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0093] It will be readily appreciated by the skilled artisan that apolypeptide and/or polynucleotide of the present invention may also beused in a method for the structure-based design of an agonist orantagonist of the polypeptide and/or polynucleotide, by: (a) determiningin the first instance the three-dimensional structure of the polypeptideand/or polynucleotide, or complexes thereof; (b) deducing thethree-dimensional structure for the likely reactive site(s), bindingsite(s) or motif(s) of an agonist or antagonist; (c) synthesizingcandidate compounds that are predicted to bind to or react with thededuced binding site(s), reactive site(s), and/or motif(s); and (d)testing whether the candidate compounds are indeed agonists orantagonists.

[0094] It will be further appreciated that this will normally be aniterative process, and this iterative process may be performed usingautomated and computer-controlled steps.

[0095] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, a Disease, relatedto either an excess of, an under-expression of, an elevated activity of,or a decreased activity of nadE polypeptide and/or polynucleotide.

[0096] If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the nadEpolypeptide and/or polypeptide.

[0097] In still another approach, expression of the gene encodingendogenous nadE polypeptide can be inhibited using expression blockingtechniques. This blocking may be targeted against any step in geneexpression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0098] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0099] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial nadE proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

[0100] In accordance with yet another aspect of the invention, there areprovided nadE agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

[0101] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0102]Helicobacter pylori herein “H. pylori”) bacteria infect thestomachs of over one-third of the world's population causing stomachcancer, ulcers, and gastritis (International Agency for Research onCancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori(International Agency for Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofnadE polypeptides and/or polynucleotides) found using screens providedby the invention, or known in the art, particularly narrow-spectrumantibiotics, should be useful in the treatment of H. pylori infection.Such treatment should decrease the advent of H. pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

[0103] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

[0104] GLOSSARY

[0105] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0106] “Bodily material(s) means any material derived from an individualor from an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials..

[0107] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

[0108] “Host cell(s)” is a cell that has been introduced (e.g.,transformed or transfected) or is capable of introduction (e.g.,transformation or transfection) by an exogenous polynucleotide sequence.

[0109] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, N.Y., 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, N.Y., 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y., 1991; andCarillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).Methods to determine identity are designed to give the largest matchbetween the sequences tested. Moreover, methods to determine identityare codified in publicly available computer programs. Computer programmethods to determine identity between two sequences include, but are notlimited to, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F.et al., J Molec. Biol 215: 403410 (1990). The BLAST X program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., JMol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithmmay also be used to determine identity.

[0110] Parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0111] Gap Penalty: 12

[0112] Gap Length Penalty: 4

[0113] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis.. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0114] Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443453 (1970)

[0115] Comparison matrix: matches=+10, mismatch=0

[0116] Gap Penalty: 50

[0117] Gap Length Penalty: 3

[0118] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0119] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0120] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO: 1 or may include up to a certain integer numberof nucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO: 1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO: 1, or:

n _(n) ≦x _(n)−(x _(n) ·Y),

[0121] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO: 1, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO: 2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

[0122] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 95, 97 or 100%identity to a polypeptide reference sequence of SEQ ID NO: 2, whereinsaid polypeptide sequence may be identical to the reference sequence ofSEQ ID NO: 2 or may include up to a certain integer number of amino acidalterations as compared to the reference sequence, wherein saidalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of amino acid alterations is determined bymultiplying the total number of amino acids in SEQ ID NO: 2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO: 2, or:

n _(a) ≦x _(a)−(x _(a) ·Y),

[0123] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO: 2, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

[0124] “Individual(s)” means a multicellular eukaryote, including, butnot limited to a metazoan, a mammal, an ovid, a bovid, a simian, aprimate, and a human.

[0125] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0126] “Organism(s)” means a (i) prokaryote, including but not limitedto, a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moracella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducrey , Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

[0127] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions.In addition, “polynucleotide” as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term “polynucleotide(s)” also includes DNAs or RNAs asdescribed above that comprise one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0128] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may compriseamino acids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may comprise many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, N.Y. (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, N.Y. (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

[0129] “Recombinant expression system(s)” refers to expression systemsor portions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

[0130] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln, and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

[0131] The examples below are carried out using standard techniques,that are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain selection, Library Production and Sequencing

[0132] The polynucleotide having a DNA sequence given in Table 1 [SEQ IDNO: 1] was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones comprising overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO: 1. Librariesmay be prepared by routine methods, for example:

[0133] Methods 1 and 2 below.

[0134] Total cellular DNA is isolated from Streptococcus pneumoniae0100993 according to standard procedures and size-fractionated by eitherof two methods.

[0135] Method 1

[0136] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E. coli infected withthe packaged library. The library is amplified by standard procedures.

[0137] Method 2

[0138] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,Bshl235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E. coliinfected with the packaged library. The library is amplified by standardprocedures.

Example 2 NadE Characterization

[0139] The determination of expression during infection of a gene fromStreptococcus pneumoniae

[0140] Excised lungs from a 48 hour respiratory tract infection ofStreptococcus pneumoniae 0100993 in the mouse is efficiently disruptedand processed in the presence of chaotropic agents and RNAase inhibitorto provide a mixture of animal and bacterial RNA. The optimal conditionsfor disruption and processing to give stable preparations and highyields of bacterial RNA are followed by the use of hybridisation to aradiolabelled oligonucleotide specific to Streptococcus pneumoniae 16SRNA on Northern blots. The RNAase free, DNAase free, DNA and proteinfree preparations of RNA obtained are suitable for Reverse TranscriptionPCR (RT-PCR) using unique primer pairs designed from the sequence ofeach gene of Streptococcus pneumoniae 0100993. Using this procedure itwas possible to demonstrate that nadE is transcibed during infection.

[0141] a) Isolation of tissue infected with Streptococcus pneumoniae0100993 from a mouse animal model of infection (lungs)

[0142]Streptococcus pneumoniae 0100993 is grown either on TSA/5% horseblood plates or in AGCH medium overnight, 37° C., 5%CO₂. Bacteria arethen collected and resuspended in phosphate-buffered saline to an A₆₀₀of approximately 0.4. Mice are anaesthetized with isofluorane and 50 mlof bacterial suspension (approximately 2×105 bacteria) is administeredintranasally using a pipetman. Mice are allowed to recover and have foodand water ad libitum. After 48 hours, the mice are euthanized by carbondioxide overdose, and lungs are aseptically removed and snap-frozen inliquid nitrogen.

[0143] b) Isolation of Streptococcus pneumoniae 0100993 RNA frominfected tissue samples

[0144] Infected tissue samples, in 2-ml cryo-strorage tubes, are removedfrom-80° C. storage into a dry ice ethanol bath. In a microbiologicalsafety cabinet the samples are disrupted up to eight at a time while theremaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroform/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at-80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

[0145] RNA preparations are stored at-80° C. for up to one month. Forlonger term storage the RNA precipitate can be stored at the wash stageof the protocol in 75% ethanol for at least one year at -20° C.

[0146] Quality of the RNA isolated is assessed by running samples on 1%agarose gels. 1× TBE gels stained with ethidium bromide are used tovisualise total RNA yields. To demonstrate the isolation of bacterialRNA from the infected tissue 1× MOPS, 2.2M formaldehyde gels are run andvacuum blotted to Hybond-N (Amersham). The blot is then hybridised witha ³²P-labelled oligonucletide probe, of sequence 5′AACTGAGACTGGCTTTAAGAGATTA 3′, specific to 16S rRNA of Streptococcuspneumoniae. The size of the hybridising band is compared to that ofcontrol RNA isolated from in vitro grown Streptococcus pneumoniae0100993 in the Northern blot. Correct sized bacterial 16S rRNA bands canbe detected in total RNA samples which show degradation of the mammalianRNA when visualised on TBE gels.

[0147] c) The removal of DNA from Streptococcus pneumoniae-derived RNA

[0148] DNA is removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

[0149] The DNAase is inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol.

[0150] DNAase treated RNA is resuspended in 100 microlitres of DEPCtreated water with the addition of Rnasin as described before.

[0151] d) The preparation of cDNA from RNA samples derived from infectedtissue

[0152] 3 microgram samples of DNAase treated RNA are reverse transcribedusing a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 150 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/×RT samples are treated withRNaseH before proceeding to the PCR reaction

[0153] e) The use of PCR to determine the presence of a bacterial cDNAspecies

[0154] PCR reactions are set up on ice in 0.2 ml tubes by adding thefollowing components: 43 microlitres PCR Master Mix (AdvancedBiotechnologies Ltd.); 1 microlitre PCR primers (optimally 18-25basepairs in length and designed to possess similar annealingtemperatures), each primer at 10 mM initial concentration; and 5microlitres cDNA.

[0155] PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600as follows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at94° C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then ahold temperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microlitre aliquots are then run out on 1%1× TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™377 Sequencer usingGeneScan™ software as supplied by Perkin Elmer).

[0156] RT/PCR controls may include +/− reverse transcriptase reactions,16S rRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Streptococcus pneumoniae 0100993 genomicsequences.

[0157] To test the efficiency of the primer pairs they are used in DNAPCR with Streptococcus pneumoniae 0100993 total DNA. PCR reactions areset up and run as described above using approx. 1 microgram of DNA inplace of the cDNA.

[0158] Primer pairs which fail to give the predicted sized product ineither DNA PCR or RT/PCR are PCR failures and as such are uninformative.Of those which give the correct size product with DNA PCR two classesare distinguished in RT/PCR: 1. Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2. Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in −RT controls

Example 3 Demonstration of gene essentiality to bacterial viability

[0159] An allelic replacement cassette was generated using PCRtechnology. The cassette consisted of a pair of 500 bp chromosomal DNAfragments flanking an erythromycin resistance gene. The chromosomal DNAsequences are the 500 bp preceding and following the DNA sequenceencoding the NDP contained in Seq. ID NO. 1

[0160] The allelic replacement cassette was introduced into S.pneumoniae R6 by transformation. Competent cells were prepared accordingto published protocols. DNA was introduced into the cells by incubationof ng quantities of allelic replacement cassette with 10⁶ cells at 30°C. for 30 minutes. The cells were transferred to 37° C. for 90 minutesto allow expression of the erythromycin resistance gene. Cells wereplated in agar containing 1 ug erythromycin per ml. Following incubationat 37° C. for 36 hours, colonies are picked and grown overnight inTodd-Hewitt broth supplemented with 0.5% yeast extract. Typically 1000transformants containing the appropriate allelic replacement areobtained. If no transformants are obtained in three separatetransformation experiments as was the case for this gene nadE , then thegene is considered as being essential in vitro

1 2 1 825 DNA Streptococcus pneumoniae CDS (1)...(823) 1 atg agt ttg caagaa acg att atc caa gag ctg ggt gtc aaa cca gtg 48 Met Ser Leu Gln GluThr Ile Ile Gln Glu Leu Gly Val Lys Pro Val 1 5 10 15 att gat gcc caggaa gaa atc cgt cgt tct att gat ttc tta aaa aga 96 Ile Asp Ala Gln GluGlu Ile Arg Arg Ser Ile Asp Phe Leu Lys Arg 20 25 30 tat ctg aaa aaa catccc ttc cta aaa acc ttt gta cta ggg att tct 144 Tyr Leu Lys Lys His ProPhe Leu Lys Thr Phe Val Leu Gly Ile Ser 35 40 45 ggg gga caa gac tca accttg gca gga cgt ttg gcg caa tta gct atg 192 Gly Gly Gln Asp Ser Thr LeuAla Gly Arg Leu Ala Gln Leu Ala Met 50 55 60 gaa gaa ctg cga gct gaa acggga gac gat agc tac aaa ttt atc gct 240 Glu Glu Leu Arg Ala Glu Thr GlyAsp Asp Ser Tyr Lys Phe Ile Ala 65 70 75 80 gtc cgc ctg cca tac gga gtgcaa gct gat gaa gca gat gct caa aaa 288 Val Arg Leu Pro Tyr Gly Val GlnAla Asp Glu Ala Asp Ala Gln Lys 85 90 95 gcc cta gcc ttc atc cag cca gatgtc agc ttg gtt gtg aat atc aag 336 Ala Leu Ala Phe Ile Gln Pro Asp ValSer Leu Val Val Asn Ile Lys 100 105 110 gaa tca gct gat gcc atg aca gctgca gtt gaa gcg aca ggt agt cct 384 Glu Ser Ala Asp Ala Met Thr Ala AlaVal Glu Ala Thr Gly Ser Pro 115 120 125 gtt tca gac ttc aac aag ggg aatatc aag gca cgt tgc cgt atg att 432 Val Ser Asp Phe Asn Lys Gly Asn IleLys Ala Arg Cys Arg Met Ile 130 135 140 gct cag tat gcc ctt gct ggt tcccat agc gga gcg gtc att gga aca 480 Ala Gln Tyr Ala Leu Ala Gly Ser HisSer Gly Ala Val Ile Gly Thr 145 150 155 160 gac cac gcc gca gaa aat atcaca ggt ttc ttt acc aag ttt ggt gac 528 Asp His Ala Ala Glu Asn Ile ThrGly Phe Phe Thr Lys Phe Gly Asp 165 170 175 ggc ggt gcg gat att ctc cctctt tac cgc ctc aat aaa cgc caa gga 576 Gly Gly Ala Asp Ile Leu Pro LeuTyr Arg Leu Asn Lys Arg Gln Gly 180 185 190 aaa cag ctc ttg cag aaa cttggc gca gag cca gcc ctt tat gaa aaa 624 Lys Gln Leu Leu Gln Lys Leu GlyAla Glu Pro Ala Leu Tyr Glu Lys 195 200 205 atc cca acg gca gac cta gaagaa gat aaa cca ggc cta gct gac gaa 672 Ile Pro Thr Ala Asp Leu Glu GluAsp Lys Pro Gly Leu Ala Asp Glu 210 215 220 gtc gca ctt gga gtc acc tacgca gag att gac gac tac cta gaa ggc 720 Val Ala Leu Gly Val Thr Tyr AlaGlu Ile Asp Asp Tyr Leu Glu Gly 225 230 235 240 aaa aca atc agc cca gaagct caa gcg acc att gaa aac tgg tgg cac 768 Lys Thr Ile Ser Pro Glu AlaGln Ala Thr Ile Glu Asn Trp Trp His 245 250 255 aaa ggc caa cac aaa cgccac tta ccc atc acc gta ttt gat gac ttt 816 Lys Gly Gln His Lys Arg HisLeu Pro Ile Thr Val Phe Asp Asp Phe 260 265 270 tgg gag t aa 825 Trp Glu2 274 PRT Streptococcus pneumoniae 2 Met Ser Leu Gln Glu Thr Ile Ile GlnGlu Leu Gly Val Lys Pro Val 1 5 10 15 Ile Asp Ala Gln Glu Glu Ile ArgArg Ser Ile Asp Phe Leu Lys Arg 20 25 30 Tyr Leu Lys Lys His Pro Phe LeuLys Thr Phe Val Leu Gly Ile Ser 35 40 45 Gly Gly Gln Asp Ser Thr Leu AlaGly Arg Leu Ala Gln Leu Ala Met 50 55 60 Glu Glu Leu Arg Ala Glu Thr GlyAsp Asp Ser Tyr Lys Phe Ile Ala 65 70 75 80 Val Arg Leu Pro Tyr Gly ValGln Ala Asp Glu Ala Asp Ala Gln Lys 85 90 95 Ala Leu Ala Phe Ile Gln ProAsp Val Ser Leu Val Val Asn Ile Lys 100 105 110 Glu Ser Ala Asp Ala MetThr Ala Ala Val Glu Ala Thr Gly Ser Pro 115 120 125 Val Ser Asp Phe AsnLys Gly Asn Ile Lys Ala Arg Cys Arg Met Ile 130 135 140 Ala Gln Tyr AlaLeu Ala Gly Ser His Ser Gly Ala Val Ile Gly Thr 145 150 155 160 Asp HisAla Ala Glu Asn Ile Thr Gly Phe Phe Thr Lys Phe Gly Asp 165 170 175 GlyGly Ala Asp Ile Leu Pro Leu Tyr Arg Leu Asn Lys Arg Gln Gly 180 185 190Lys Gln Leu Leu Gln Lys Leu Gly Ala Glu Pro Ala Leu Tyr Glu Lys 195 200205 Ile Pro Thr Ala Asp Leu Glu Glu Asp Lys Pro Gly Leu Ala Asp Glu 210215 220 Val Ala Leu Gly Val Thr Tyr Ala Glu Ile Asp Asp Tyr Leu Glu Gly225 230 235 240 Lys Thr Ile Ser Pro Glu Ala Gln Ala Thr Ile Glu Asn TrpTrp His 245 250 255 Lys Gly Gln His Lys Arg His Leu Pro Ile Thr Val PheAsp Asp Phe 260 265 270 Trp Glu

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidhaving at least 95% identity to the amino acid sequence of SEQ ID NO: 2over the entire length of SEQ ID NO: 2; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 2, (iii) an isolatedpolypeptide that is the amino acid sequence of SEQ ID NO: 2, and (iv) apolypeptide that is encoded by a recombinant polynucleotide comprisingthe polyncleotide sequence of SEQ ID NO:
 1. 2. An isolatedpolynucleotide selected from the group consisting of: (i) an isolatedpolynucleotide comprising a polynucleotide sequence encoding apolypeptide that has at least 95% identity to the amino acid sequence ofSEQ ID NO: 2, over the entire length of SEQ ID No: 2; (ii) an isolatedpolynucleotide comprising a polynucleotide sequence that has at least95% identity over its entire length to a polynucleotide sequenceencoding the polypeptide of SEQ ID NO: 2; (iii) an isolatedpolynucleotide comprising a nucleotide sequence that has at least 95%identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1;(iv) an isolated polynucleotide comprising a nucleotide sequenceencoding the polypeptide of SEQ ID NO: 2; (v) an isolated polynucleotidethat is the polynucleotide of SEQ ID NO: 1; (vi) an isolatedpolynucleotide of at least 30 nucleotides in length obtainable byscreening an appropriate library under stringent hybridizationconditions with a probe having the sequence of SEQ ID NO: 1 or afragment thereof of of at least 30 nucleotides in length; (vii) anisolated polynucleotide encoding a mature polypeptide expressed by thenadE gene comprised in the streptococcus pneumoniae; and (viii) apolynucleotide sequence complementary to said isolated polynucleotide of(i), (ii), (iii), (iv), (v), (vi) or (vii).
 3. A method for thetreatment of an individual: (i) in need of enhanced activity orexpression of or immunological response to the polypeptide of claim 1comprising the step of: administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or (ii) having need to inhibit activity or expression of the polypeptideof claim 1 comprising: (a) administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or b) administering to the individual a nucleic acid molecule thatinhibits the expression of a polynucleotide sequence encoding saidpolypeptide; (c) administering to the individual a therapeuticallyeffective amount of a polypeptide that competes with said polypeptidefor its ligand, substrate, or receptor; or (d) administering to theindividual an amount of a polypeptide that induces an immunologicalresponse to said polypeptide in said individual.
 4. A process fordiagnosing or prognosing a disease or a susceptibility to a disease inan individual related to expression or activity of the polypeptide ofclaim 1 in an individual comprising the step of: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid polypeptide in an organism in said individual; or (b) analyzing forthe presence or amount of said polypeptide expression in a samplederived from said individual.
 5. A process for producing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO: 2 over theentire length of SEQ ID NO: 2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO: 2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO: 1, comprising the step ofculturing a host cell under conditions sufficient for the production ofthe polypeptide.
 6. A process for producing a host cell comprising anexpression system or a membrane thereof expressing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO: 2 over theentire length of SEQ ID NO: 2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO: 2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO: 1, said process comprising thestep of transforming or transfecting a cell with an expression systemcomprising a polynucleotide capable of producing said polypeptide of(i), (ii), (iii) or (iv) when said expression system is present in acompatible host cell such the host cell, under appropriate cultureconditions, produces said polypeptide of (i), (ii), (iii) or (iv).
 7. Ahost cell or a membrane expressing a polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least 95% identity to theamino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 2; (iii) an isolated polypeptide that is the amino acidsequence of SEQ ID NO: 2, and (iv) a polypeptide that is encoded by arecombinant polynucleotide comprising the polynucleotide sequence of SEQID NO:
 1. 8. An antibody immunospecific for the polypeptide of claim 1.9. A method for screening to identify compounds that agonize or thatinhibit the function of the polypeptide of claim 1 that comprises amethod selected from the group consisting of (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution comprising a polypeptide of claim 1,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 10. An agonist or antagonist to thepolypeptide of claim 1.